Enhancement Of Epithelial-mesenchymal Transition Research Articles

Hypoxia enhances motility and EMT through the Na+/H+ exchanger NHE-1 in MDA-MB-231 breast cancer cells

Breast cancer metastasis is the leading cause of cancer-related deaths. Hypoxia in the tumor mass is believed to trigger cell migration, which is involved in a crucial process of breast cancer metastasis. However, the molecular mechanisms underlying aggressive behavior under hypoxic conditions have not been fully elucidated. Here, we demonstrate the significant motility of MDA-MB-231 cells cultured under hypoxic conditions compared to that of cells cultured under normoxic conditions. MDA-MB-231 cells under hypoxic conditions showed a significant increase in Na+/H+ exchanger isoform 1 (NHE1) expression level, which was observed to co-locate in lamellipodia formation. Inhibition of NHE1 significantly suppressed the intracellular pH and the expression of mesenchymal markers, thereby blocking the high migration activity in hypoxia. Moreover, treatment with ciglitazone, a potent and selective peroxisome proliferator-activated receptor γ (PPARγ) agonist, modulated hypoxia-enhanced motion in cells via the repression of NHE1. These findings highlight that NHE1 is required for migratory activity through the enhancement of epithelial-mesenchymal transition (EMT) in MDA-MB-231 cells under hypoxic conditions, and we propose new drug repurposing strategies targeting hypoxia based on NHE1 suppression by effective usage of PPARγ agonists.

Activation of PGK1 under hypoxic conditions promotes glycolysis and increases stem cell‑like properties and the epithelial‑mesenchymal transition in oral squamous cell carcinoma cells via the AKT signalling pathway.

Although it has been previously documented that a hypoxic environment can promote glycolysis and the malignant progression of oral squamous cell carcinoma (OSCC) cells, the specific underlying mechanism remains unclear. Phosphoglycerate kinase 1 (PGK1) has been previously reported to serve an important role in tumor metabolism. The aim of the present study was to investigate the effects of hypoxia and PGK1 on glycolysis, stem cell-like properties and epithelial-mesenchymal transition (EMT) in OSCC cells. Cell Counting Kit-8 assays were performed to examine tumor cell viability under hypoxic conditions. Sphere formation, immunohistochemistry, western blotting, Transwell assays and mouse xenograft studies were performed to assess the biological effects of PGK1. Under hypoxic conditions, phosphoglycerate PGK1 expression was found to be upregulated, which resulted in the potentiation of stem cell-like properties and enhancement of EMT. However, PGK1 knockdown reversed hypoxia-mediated glycolysis, stem cell-like properties, EMT in addition to inhibiting OSCC cell invasion and migration. PGK1 knockdown also inhibited tumour growth, whilst the overexpression of PGK1 was demonstrated to promote tumour growth in mouse xenograft models in vivo. Downstream, activation of the AKT signalling pathway reversed the series of changes induced by PGK1 knockdown. PGK1 expression was found to be upregulated in human OSCC tissues, which was associated with the pathological differentiation of tumours and lymph node metastasis. To conclude, results from the present study demonstrate that hypoxia can increase PGK1 expression, resulting in the promotion of glycolysis, enhancing stem cell-like properties and EMT by activating AKT signalling in OSCC.

Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder.

Disabled homolog-2 (DAB2) has been reported to be a tumor suppressor gene. However, a number of contrary studies suggested that DAB2 promotes tumor invasion in urothelial carcinoma of the bladder (UCB). Here, we investigated the clinical role and biological function of DAB2 in human UCB. Immunohistochemical staining analysis for DAB2 was carried out on UCB tissue specimens. DAB2 expression levels were compared with clinicopathological factors. DAB2 was knocked-down by small interfering RNA (siRNA) transfection, and then its effects on cell proliferation, invasion, and migration, and changes to epithelial-mesenchymal transition (EMT)-related proteins were evaluated. In our in vivo assays, tumor-bearing athymic nude mice subcutaneously inoculated with human UCB cells (MGH-U-3 or UM-UC-3) were treated by DAB2-targeting siRNA. Higher expression of DAB2 was associated with higher clinical T category, high tumor grade, and poor oncological outcome. The knock-down of DAB2 decreased both invasion and migration ability and expression of EMT-related proteins. Significant inhibitory effects on tumor growth and invasion were observed in xenograft tumors of UM-UC-3 treated by DAB2-targeting siRNA. Our findings suggested that DAB2 expression was associated with poor prognosis through increased oncogenic properties including tumor proliferation, migration, invasion, and enhancement of EMT in human UCB.

Gonadotropins promote human ovarian cancer cell migration and invasion via a cyclooxygenase 2-dependent pathway.

It is generally accepted that ovarian cancer is associated with local elevation of gonadotropins (FSH and LH), with repeated ovulation and accompanying expression of inducible cyclooxygenase2 (COX2). However, the roles of gonadotropins and the concomitant elevation of COX2 in the development of ovarian cancer have not been fully characterized. Herein, we report that excessive FSH/LH exposure did not induce proliferation in ovarian cancer cell lines but significantly promoted cell migration and invasion. Moreover, FSH/LH treatment rapidly upregulated COX2 expression within 24h, whereas COX1 expression remained unchanged. Further results showed that enhancement of epithelial-mesenchymal transition (EMT) and upregulation of matrix metalloproteinase (MMP)2 and MMP9 contributed to the stimulatory effect of gonadotropins on cell migration and invasion; these effects were sufficiently blocked by a selective COX2 inhibitor. In conclusion, the present study suggests that gonadotropin-induced migration and invasion in ovarian cancer may be caused by EMT and MMP upregulation via a COX2-dependent pathway.

VEGF/NRP-1axis promotes progression of breast cancer via enhancement of epithelial-mesenchymal transition and activation of NF-κB and β-catenin

Autocrine vascular endothelial growth factor (VEGF) can regulate the survival and progression of cancers through its various receptors. But the mechanisms and mediators for these functions are largely uncovered, especially in breast cancer. We examined the potential roles and mechanisms of VEGF/neuropilin-1 (NRP-1) axis in regulating the tumorigenesis and metastasis of breast cancer and found the expression of VEGF and NRP-1 correlated with aggressiveness of breast cancer. Knockdown of VEGF or NRP-1 inhibited the proliferation, migration and invasion, but enhanced the apoptosis of MDA-MB-231 cells. In contrast, induction of NRP-1 over-expression promoted the proliferation, migration and invasion of MCF-7 cells. VEGF or NRP-1 silencing attenuated the epithelial-mesenchymal transition (EMT) process and the activation of NF-κBp65, but enhanced GSK-3β expression in MDA-MB-231 cells while NRP-1 over-expression reversed the effects in MCF-7 cells. Treatment with hVEGF165 did not change the inhibition in NRP-1 silencing MDA-MB-231 cells, but enhanced the aggressiveness of NRP-1 over-expressing MCF-7 cells. In addition, VEGF-silencing inhibited the growth and metastasis of implanted MDA-MB-231 tumors in vivo. Our novel data suggest that the positive regulation of the VEGF/NRP-1 axis on the tumorigenesis and metastasis of breast cancer may be associated with enhancing the EMT process and the NF-κB and β-catenin signaling. Hence, the VEGF/NRP-1 axis may be a valuable target for design of therapies for intervention of breast cancer.

Tumor necrosis factor superfamily member LIGHT induces epithelial–mesenchymal transition in A549 human alveolar epithelial cells

Fibrosis is an abnormal response to organ injury, characterized by accumulation of activated fibroblasts at the sites of injury. Fibroblasts arise from several sources, including resident fibroblasts and circulating fibrocytes that infiltrate organ tissue. Recently, epithelial–mesenchymal transition (EMT) has been recognized as a source of mesenchymal cells. EMT is induced by various growth factors, such as transforming growth factor (TGF)-β1, and enhanced by inflammatory cytokines. Recently the tumor necrosis factor superfamily member LIGHT has been implicated in the pathogenesis of inflammatory disease and airway remodeling in severe asthma. We hypothesized that LIGHT might contribute to the pathogenesis of airway fibrosis via enhancement of EMT. Therefore, we investigated LIGHT’s ability to induce EMT. A549 cells were stimulated with LIGHT, TGF-β1 or both for 48h. To estimate EMT, we evaluated the expression of epithelial and mesenchymal markers using immunocytochemistry, Western blotting and quantitative RT-PCR. Signaling pathways for EMT were characterized by Western analysis to detect phosphorylation of Erk1/2 and smad2. LIGHT enhanced TGF-β1-induced EMT both morphologically, by suppressing E-cadherin and enhancing vimentin, and functionally, by enhancing cell contractility. Additionally, LIGHT induced EMT without TGF-β1. Evaluation of the mechanism showed that LIGHT did not induce TGF-β1 production or affect the smad-snai1 pathway. Inhibition of Erk1/2 phosphorylation reduced LIGHT-induced EMT, indicating the Erk1/2 pathway to be a key pathway in LIGHT-induced EMT. In summary, LIGHT enhanced TGF-β1-induced EMT but also induced EMT via the Erk1/2 pathway by itself, without TGF-β1 signaling. LIGHT may contribute to the pathogenesis of airway fibrosis through enhancement of EMT.

Heavy Ions Can Enhance TGFβ Mediated Epithelial to Mesenchymal Transition

TGFβ is a key modulator of the Epithelial-Mesenchymal Transition (EMT), a process important in cancer progression and metastasis, which leads to the suppression of epithelial genes and expression of mesenchymal proteins. Ionizing radiation was found to specifically induce expression of the TGF-β1 isoform, which can modulate late post-radiation changes and increase the risk of tumor development and metastasis. Interactions between TGFβ induced EMT and DNA damage responses have not been fully elucidated, particularly at low doses and following different radiation quality exposures. Further characterization of the relationship between radiation quality, EMT and cancer development is warranted. We investigated whether space radiation induced TGFβ dependent EMT, using hTERT immortalized human esophageal epithelial cells (EPC2-hTERT) and non-transformed mink lung epithelial cells (Mv1Lu). We have observed morphologic and molecular alterations in EPC2 and Mv1Lu cells consistent with EMT after pre-treatment with TGFβ1. This effect could be efficiently inhibited in both cell lines by the use of a TGFβRI inhibitor. High-energy silicon or iron nuclei were each able to cause a mild induction of EMT, with the inclusion of TGFβ1 inducing a greatly enhanced EMT phenotype even when cells were irradiated with doses as low as 0.1 Gy. A further enhancement of EMT was achieved at a higher dose of 2 Gy. TGFβRI inhibitor was able to reverse the EMT induced by the combination of TGFβ1 and radiation. These studies indicate that heavy ions, even at a low dose, may trigger the process of TGFβ1-induced EMT, and suggest further studies are needed to determine whether the chronic exposures received in space may potentiate this process in astronauts, leading to an increased risk of cancer.

CD133/Src Axis Mediates Tumor Initiating Property and Epithelial-Mesenchymal Transition of Head and Neck Cancer

BackgroundHead and Neck squamous cell carcinoma (HNSCC) is a human lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. Caner initiating cells (CICs), which are responsible for tumor growth and coupled with gain of epithelial-mesenchymal transition (EMT), have been identified. Previously, we enriched a subpopulation of head and neck cancer initiating cells (HN-CICs) with up-regulation of CD133 and enhancement of EMT. Others demonstrate that Src kinase interacts with and phosphorylates the cytoplasmic domain of CD133. However, the physiological function of CD133/Src signaling in HNSCCs has not been uncovered.Methodology/Principal FindingHerein, we determined the critical role of CD133/Src axis modulating stemness, EMT and tumorigenicity of HNSCC and HN-CICs. Initially, down-regulation of CD133 significantly reduced the self-renewal ability and expression of stemness genes, and promoted the differentiation and apoptotic capability of HN-CICs. Additionally, knockdown of CD133 in HN-CICs also lessened both in vitro malignant properties including cell migration/cell invasiveness/anchorage independent growth, and in vivo tumor growth by nude mice xenotransplantation assay. In opposite, overexpression of CD133 enhanced the stemness properties and tumorigenic ability of HNSCCs. Lastly, up-regulation of CD133 increased phosphorylation of Src coupled with EMT transformation in HNSCCs, on the contrary, silence of CD133 or treatment of Src inhibitor inversely abrogated above phenotypic effects, which were induced by CD133 up-regulation in HNSCCs or HN-CICs.Conclusion/SignificanceOur results suggested that CD133/Src signaling is a regulatory switch to gain of EMT and of stemness properties in HNSCC. Finally, CD133/Src axis might be a potential therapeutic target for HNSCC by eliminating HN-CICs.

Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

Epithelial-mesenchymal transition (EMT) is a key step toward cancer metastasis, and Snail is a major transcription factor governing EMT. Here, we demonstrate that Snail-induced EMT accelerates cancer metastasis through not only enhanced invasion but also induction of immunosuppression. Murine and human melanoma cells with typical EMT features after snail transduction induced regulatory T cells and impaired dendritic cells in vitro and in vivo partly through TSP1 production. Although Snail(+) melanoma did not respond to immunotherapy, intratumoral injection with snail-specific siRNA or anti-TSP1 monoclonal antibody significantly inhibited tumor growth and metastasis following increase of tumor-specific tumor-infiltrating lymphocytes and systemic immune responses. These results suggest that inhibition of Snail-induced EMT could simultaneously suppress both tumor metastasis and immunosuppression in cancer patients.